(A) F9 cells were treated with vehicle or Akti-1/2 for 2 days, cross-linked with formaldehyde, and immunoprecipitated with anti-Oct4. The immunocomplexes were subjected to immunoblotting with anti-Oct4 (upper) or anti-Sox2 (lower). (B) Enhanced interaction of ectopically expressed Flag-Oct4-T235D with endogenous Sox2 in NCCIT cells. (C) Akt-mediated phosphorylation of Oct4 enhances its binding with Sox2 and Klf4 in vitro. (D) Akt-mediated phosphorylation of Oct4 enhances its binding to the promoters of three key stemness genes in vitro. The arrow indicates the Oct4-Sox2-probe complexes. (E) Akt-mediated phosphorylation enhances Oct4 transcriptional activity in a reporter assay. (F) NCCIT cells were treated with vehicle or Akti-1/2 for 3 days and subjected to ChIP-based analysis. (G) NCCIT cells were treated with vehicle or Akti-1/2 for 3 days, and the transcription levels of POU5F1, NANOG and SOX2 determined by qRT-PCR. (H) NCCIT cells were treated in the same manner as (G) and samples subjected to immunoblotting. The error bars in the (E), (F) and (G) represent mean ± SD from three independent experiments. * and *** denotes P < 0.05 and P < 0.001, respectively.